Fig 2: Generation of SMART Tg mice.a Depiction of the SMART biosensor structure (top) and its activation mechanism (bottom). SMART is composed of N-terminal Ypet, a modified α1 to α4 helices of the KL domain of MLKL, and C-terminal ECFP. Upon necroptosis induction, activated and phosphorylated RIPK3 (pRIPK3) phosphorylates MLKL, resulting in oligomer formation of MLKL. Then, oligomers of MLKL induces conformational changes of SMART, possibly through the interaction, thereby increasing FRET efficiency. KL pseudokinase domain, Ypet modified yellow fluorescent protein, ECFP enhanced cyan fluorescent protein, pRIPK3 phosphorylated RIPK3. b Western blots probed with anti-GFP antibody show expression of the SMART biosensor in various murine tissues. Tissue extracts were prepared from the indicated organs of 8-week-old wild-type or SMART Tg mice. Results are representative of two independent experiments. c Mice were intraperitoneally injected with thioglycollate, then peritoneal cells were recovered by washing the peritoneal cavity with ice-cold PBS on day 4 after injection. Isolated cells were stained with the indicated antibodies and analyzed with flow cytometry. The percentages of CD11b+F4/80+ cells indicate the fraction of macrophages; the levels of YFP detected in these cell populations indicate the expression of SMART. Results are representative of three independent experiments. WT wild-type. d Peritoneal macrophages from SMART Tg mice were untreated or stimulated with BV6 (1 μM) + zVAD (20 μM) or BV6 (1 μM) + zVAD (20 μM) + GSK’872 (5 μM) for the indicated times. Cell death was assessed with the LDH release assay. Results are mean ± SD of triplicate samples, and they are representative of five independent experiments. e, f Peritoneal macrophages derived from SMART Tg mice were stimulated as described in d, and FRET/CFP ratios were calculated. Pseudocolored images show cellular changes in FRET/CFP ratio values in response to the indicated stimulations (e). FRET/CFP responses are color-coded according to the color scales (right). White arrowheads indicate cells undergoing necroptosis. Scale bars, 20 μm. Maximum changes detected in the FRET/CFP ratios (f). Results are mean ± SE (n = 11 cells per condition). Each dot indicates an individual cell. Results are representative of four independent experiments. Statistical significance was determined with two-way ANOVA with Dunnett’s multiple comparison test (d) or one-way ANOVA with Turkey’s multiple comparison test (f).

Fig 3: Crucial role for RIPK3 and MLKL in the FRET response of SMART in macrophages following necroptosis induction.a Peritoneal macrophages from SMART WT, SMART Ripk3−/−, SMART Mlkl−/− mice were untreated or stimulated with BV6 (1 μM) + zVAD (20 μM) or BV6 (1 μM) + zVAD (20 μM) + GSK’872 (5 μM) for the indicated periods. Cell death was determined with the LDH release assay. Results are mean ± SD of triplicate samples. b, c Peritoneal macrophages were stimulated and analyzed as described in a. Pseudocolored images show cellular changes in FRET/CFP ratio values in response to the indicated stimulations (b). FRET/CFP responses are color-coded according to the color scales (right). White arrowheads indicate cells undergoing necroptosis. Scale bars, 20 μm. Maximum changes detected in the FRET/CFP ratio (c). Results are mean ± SE (n = 10 cells). Each dot indicates an individual cell. Statistical analysis was performed with two-way ANOVA with Dunnett’s multiple comparison test (a, left panel) or Sidak’s multiple comparison test (a, middle and right panels), one-way ANOVA with Tukey’s multiple comparison test (c, left panel), or the unpaired two-tailed Student t-test (c, middle and right panels). All results are representative of three independent experiments.

Fig 4: Administration of cisplatin induces necroptosis in renal proximal tubular cells.a–f Eight-week-old wild-type were intravenously injected with cisplatin (20 mg/kg), then sacrificed at the indicated times. Concentrations of blood urea nitrogen (BUN) and serum creatinine were determined at the indicated times after cisplatin treatment (a). Results are mean ± SE (n = 3 mice per time point). Results are representative of two independent experiments. Kidney tissue sections were prepared at the indicated times after cisplatin treatment and stained with hematoxylin & eosin (n = 5 mice) (b). Scale bar, 50 μm. Asterisks indicate dilated proximal tubules. Kidney tissue sections were stained with anti-phospho-RIPK3 (pRIPK3) (c) or anti-cleaved caspase 3 (CC3) (d) antibodies. White arrowheads and black arrows indicate pRIPK3+ and CC3+ cells, respectively. Scale bars, 50 μm. Cell counts for pRIPK3+ (e) and CC3+ (f) cells, expressed as the number of positive cells per field. Results are mean ± SE (n = 5 mice). g–j Eight-week-old wild-type and Ripk3−/− mice were injected with cisplatin as in a and sacrificed on day 2 after injection (wild-type, n = 5 mice; Ripk3−/− mice, n = 6 mice). Concentrations of BUN and serum creatinine were determined as in a (g). Kidney tissue sections were stained with anti-pRIPK3 (h) or anti-CC3 (i) antibodies. Scale bars, 50 μm. The numbers of CC3+ cells were counted as in f (j). White arrowheads and black arrows indicate pRIPK3+ and CC3+ cells, respectively. Statistical analyses were performed with one-way ANOVA and Dunnett’s multiple comparison test (a, e, f) or the unpaired two-tailed Student t-test (g, j).